Notes

Abstract:

Standard CPR (S-CPR) techniques for children are inadequate. Survival rates are poor for in-hospital and out-of-hospital cardiac arrest. Rescuers rarely achieve the appropriate compression or decompression during CPR. We created a novel adhesive glove device (AGD) to aid in active compression-decompression pediatric CPR. New CPR devices must improve blood flow. In animal CPR models, microspheres are injected into the left ventricle while a reference blood sample is withdrawn from the descending aorta. Tissue samples are obtained for organ analysis. Using a validated formula, researchers calculate regional blood flow. Non-radioactive neutron-activated microspheres have replaced more dangerous radioactive microspheres originally used. Neurological outcome in survivors is critical. Recently, researchers found biomarkers in the cerebral spinal fluid and plasma released from the breakdown of nerve cells. Biomarker levels may correlate with the degree of neurological insult. UCHL1 is released upon the breakdown of perikarya and dendrites. Alpha-synuclein is a presynaptic protein. pNF-H is released upon injury to axons. Methods.Thirty-one farm piglets were sedated, anesthetized, and cannulated. Baseline measurements were obtained. Ventricular fibrillation (VF) was induced by direct current. After 3 to 5 minutes of untreated VF, 12 animals received S-CPR and 19 received AGD-CPR. Six animals in each group received microsphere injections (BioPAL, Inc, Worcester, MA) for blood flow analyses. Plasma for biomarkers (EnCor Biotech, Gainesville, FL) were collected at baseline, VF, after 2 minutes of CPR, at the return of spontaneous circulation (ROSC), 30 minutes after ROSC, and before euthanasia. We collected specimens for microsphere analyses at necropsy. Results.Blood gas and electrolyte parameters as well as blood flow to the brain, heart apex, heart septum, kidney, and liver were similar between S-CPR and AGD-CPR groups at baseline and ROSC. UCHL1, pNF-H, and alpha-syn did not show interaction between time and CPR group. Mean alpha-syn levels in the S-CPR group at baseline were higher than the AGD-CPR group (P=0.05), but both groups had large standard deviations. Conclusion. Our microsphere blood flow technique was flawed. Blood draws for microsphere analyses were performed too early after ischemic insult and did not show a significant change. Refinement of this approach is necessary.

General Note:

In the series University of Florida Digital Collections.

General Note:

Includes vita.

Bibliography:

Includes bibliographical references.

Source of Description:

Description based on online resource; title from PDF title page.

Source of Description:

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Notes

Abstract:

Standard CPR (S-CPR) techniques for children are inadequate. Survival rates are poor for in-hospital and out-of-hospital cardiac arrest. Rescuers rarely achieve the appropriate compression or decompression during CPR. We created a novel adhesive glove device (AGD) to aid in active compression-decompression pediatric CPR. New CPR devices must improve blood flow. In animal CPR models, microspheres are injected into the left ventricle while a reference blood sample is withdrawn from the descending aorta. Tissue samples are obtained for organ analysis. Using a validated formula, researchers calculate regional blood flow. Non-radioactive neutron-activated microspheres have replaced more dangerous radioactive microspheres originally used. Neurological outcome in survivors is critical. Recently, researchers found biomarkers in the cerebral spinal fluid and plasma released from the breakdown of nerve cells. Biomarker levels may correlate with the degree of neurological insult. UCHL1 is released upon the breakdown of perikarya and dendrites. Alpha-synuclein is a presynaptic protein. pNF-H is released upon injury to axons. Methods.Thirty-one farm piglets were sedated, anesthetized, and cannulated. Baseline measurements were obtained. Ventricular fibrillation (VF) was induced by direct current. After 3 to 5 minutes of untreated VF, 12 animals received S-CPR and 19 received AGD-CPR. Six animals in each group received microsphere injections (BioPAL, Inc, Worcester, MA) for blood flow analyses. Plasma for biomarkers (EnCor Biotech, Gainesville, FL) were collected at baseline, VF, after 2 minutes of CPR, at the return of spontaneous circulation (ROSC), 30 minutes after ROSC, and before euthanasia. We collected specimens for microsphere analyses at necropsy. Results.Blood gas and electrolyte parameters as well as blood flow to the brain, heart apex, heart septum, kidney, and liver were similar between S-CPR and AGD-CPR groups at baseline and ROSC. UCHL1, pNF-H, and alpha-syn did not show interaction between time and CPR group. Mean alpha-syn levels in the S-CPR group at baseline were higher than the AGD-CPR group (P=0.05), but both groups had large standard deviations. Conclusion. Our microsphere blood flow technique was flawed. Blood draws for microsphere analyses were performed too early after ischemic insult and did not show a significant change. Refinement of this approach is necessary.

General Note:

In the series University of Florida Digital Collections.

General Note:

Includes vita.

Bibliography:

Includes bibliographical references.

Source of Description:

Description based on online resource; title from PDF title page.

Source of Description:

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Statement of Responsibility:

by Melissa A Lamb.

Thesis:

Thesis (M.S.)--University of Florida, 2012.

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Adviser: Byrne, Barry J.

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UFE0044820:00001

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1 NEUTRON ACTIVATED MICROSPHERES AND BIOMARKERS IN A PIGLET MODEL OF CARDIAC ARREST USING A NOVEL ADHESIVE GLOVE DEVICE By MELISSA A LAMB A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Melissa A. Lamb

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3 To my parents, siblings, in laws, and grandmother, your unending support and encouragement were essential to my success. To my husband, I love you so much. Thank you for your presence, expertise, and excellent Daddy skills. I cannot imagine having a better mentor!

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4 ACKNOWLEDGMENTS I thank my colleagues in the University of Florida Pediatric Critical Care Division for the ir understanding and support. Also, I thank Dr. Richard Snyder for always looking out for my best interest as a student.

11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NEUTRON ACTIVATED MICROSPHERES AND BIOMARKERS IN A PIGLET MODEL OF CARDIAC ARREST USING A NOVEL ADHESIVE GLOVE DEVIC E By Melissa A Lamb December 2012 Chair: Barry Byrne Major: Medical Sciences Standard CPR (S CPR) techniques for children are inadequate. Survival rates are poor for in hospital and out of hospital cardiac arrest. Rescuers rarely achieve the appropriate compression or decompression during CPR. We created a novel adhesive glove d evice (AGD) to aid in active compression decompression pediatric CPR. New CPR devices must improve blood flow. In animal CPR models, microspheres are injected into the left ventricle while a reference blood sample is withdrawn from the descending aorta. Tissue samples are obtained for organ analysis. Using a validated formula, researchers calculate regional blood flow. Non radioactive neutron activated microspheres have replaced more dangerous radioactive microspheres originally used. N eurological outc ome in surviv ors is critical. Recently researchers found biomarkers in the cerebral spinal fluid and plasma released from the breakdown of nerve cells. Biomarker levels may correlate with the degree of neurological insult. UCHL1 is released upon the br eakdown of perikarya and dendrites. Alpha syn uclein is a presynaptic protein. pNF H is released upon injury to axons. Methods. Thirty one farm piglets were sedated, anesthetized, and cannulated. Baseline measurements were obtained. Ventricular fibrill ation (VF) was induced by

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12 direct current. After 3 to 5 minutes of untreated VF, 12 animals received S CPR and 19 received AGD CPR. Six animals in each group received microsphere injections (BioPAL, Inc, Worcester, MA) for blood flow analyses. Plasma for biomarkers (EnCor Biotech, Gainesville, FL) were collected at baseline, VF, after 2 minutes of CPR, at the return of spontaneous circulation (ROSC), 30 minutes after ROSC, and before euthanasia. We collect ed specimens for microsphere analyses at necropsy Results. Blood gas and electrolyte parameters as well as blood flow to the brain, heart apex, heart septum, kidney, and liver were similar between S CPR and AGD CPR groups at baseline and ROSC. UCHL1, pNF syn did not show interaction between time and CPR group. M ean syn levels in the S CPR group at baseline were higher than the AGD CPR group (P=0.05) but both groups had large standard deviations. Conclusion. Our microsphere blood flow technique was flawe d Blood draws for microsphere analyses w ere performed too early after ischemic insult and did not show a significant change. Refinement of this approach is necessary.

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13 CHAPTER 1 INTRODUCTION CPR techniques for children are largely inadequate. Only 6.4% of children survive cardiac arrest occurring outside a hospital. 1 In addition, only 4% of children experiencing cardiac arrest are neurologically in tact when they are discharged from the hospital. 2 Rescuers rarely achieve the appropriate compression depth during CPR. 3 In addition, research illustrates that effective decompression is essential for blood flow back to the heart during CPR. 4 The Food and Drug Administration has approved devices to assist rescuers in CPR, but no ne of these approved devices are designed for use on children. Children and CPR are different from that of an adult. Thus, research is necessary on new CPR devices bett er suited to infants and children. CPR device research is not without complications. The effects of a CPR device on organ perfusion are difficult to measure. Stable isotope labeled microspheres are widely accepted in other research areas for detecting or gan perfusion. 5,6 However, the use of such microspheres in a CPR model is more complex. 7 The neurological damage caused by cardiac arrest and subsequent reperfusion injuries after CPR is also difficult to predict. Researchers have validated several biomarkers that play a role in pre dicting neurological damage. 8 However, the neural system has many components, and a panel of biomarkers would best indicate future neurological outcomes. Researchers continue to discover and validate neurological biomarkers but are still trying to determine whether the levels of bio markers released into the blood correlate with the degree of neurological insult and future brain function.

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14 Hypotheses of the project: H1. AGD CPR will improve survival when compared to S CPR. H2. Neurological biomarkers will be released into the blood wit hin 30 minutes of neurological insult. H3. Non radioactive isotope microsphere technique will adequately assess blood flow to the major organs.

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15 CHAPTER 2 LITERATURE REVIEW Despite decades of research on pediatric CPR, survival rates in children are still dismal. Only 27% of children experiencing cardiac arrest while in the hospital survive to discharge. 9 Survival rates of children experiencing cardiac arrest outside of the hospital are much worse. Three percent of infants, 9 % of children and 8% of adolescents actually survive to hospital discharge when experiencing an out of hospital cardiac arrest. 1 Several factors contribute to poor survival in children experiencing out of hospital cardiac arrest. First, by standers witnessing the cardiac arrest are not performing CPR. Second, the CPR performed is inefficient and fra ught with poor technique. Third, rescuers fatigue quickly when performing CPR, adding to inefficiencies. CPR Devices Researchers have created several medical devices to remedy the inefficiencies of CPR. These medical devices focus on several different aspects of CPR. One device, the inspiratory threshold device (ITD), works to maintain negative pressure within the chest cavity. Other devices focus on the decompression phase of CPR. Many of these devices also reduce rescuer fatigue. These CPR devices are widely studied and described in detail below. I nspiratory T hreshold D evice The ITD prevents the inspiration of air during the decompression phase of CPR. When the chest is compressed, air is forced out of the chest. By impeding inspiration, a negat ive pressure is created in the thoracic cavity. The ITD creates a pressure gradient which encourages blood to flow from the extremities (positive pressure) towards the

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16 heart (negative pressure). Voelckel et al. studied the ITD with active compression dec ompression CPR (ACD received 50 Hz of alternating current via two subcutaneous needles and were left in untreated ventricular fibrillation for 10 minutes. An AMBU pump delivered Standard CPR chest compressions for eight minutes. Researchers then administered ACD+ITD CPR for an additional eight minutes using a silicon suction cup and an ITD. After a total of 26 minutes of cardiac arrest, rescuers administered 3 shocks of direct current at increasi ng levels. Six of the seven animals were resuscitated and survived for 15 minutes without medical intervention. The ACD +ITD CPR produced significantly greater blood flow to the left ventricle, cerebrum, and kidneys when compared to standard CPR (S CPR). Blood flow to the brain during ACD+ITD CPR was similar to brain blood flow prior to cardiac arrest. Coronary perfusion pressure was also significantly higher during ACD+ITD CPR than during S CPR. 10 The aforementioned researchers subsequently studied ACD+ITD CPR in adults experiencing out of hospital cardiac arrest. Subjects were randomized to either the ACD+ITD CPR intervention group or to the S CPR control group. Rescuers used a suction cup device with a handle (ResQPump) to perform ACD CPR. For subjects randomized to the ACD+ITD CPR group, rescuers placed an ITD (ResQPOD) between the bag and facemask. Rescuers were encouraged to perform CPR for at least 30 minutes in both study groups. All stud y interventions were stopped when subjects experienced a return of spontaneous circulation (ROSC) or when they arrived at the hospital. Then, hospital physicians provided traditional cardiac care. Researchers used the modified Rankin scale to quantify ne urological function at the time of hospital

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17 discharge. A score less than three was considered favorable. Subjects receiving the intervention were 53% more likely to survive to hospital discharge than subjects receiving S CPR. The rates of ROSC and hospi tal admission were similar between groups. Both groups had similar neurological outcomes and almost all survivors had no or mild neurological deficits. Remarkably, the subjects receiving ACD CPR were more likely to experience pulmonary edema, likely due to the force of the compressions administered by the ResQPump. 11 Lund University Cardiopulmonary Assist System ( LUCAS TM ) A research group in Sweden developed a gas driven CPR device, LUCAS TM LUCAS TM is composed of a rubber suction cup attached to a pneumatic cylinder that is attached to two legs. The two legs of LUCAS attach to a back plate that the patient lays on. LUCAS TM is a transportable device and is compat ible with ambulance stretchers. Steen et al. tested LUCAS TM on an artificial thorax model, a pig model, and in a clinical pilot study. The study in the thorax model showed that LUCAS TM generates instantaneous increases and decreases in pressure and main tains peak pressure, unlike S CPR. In the Group I pig model, S CPR was compared with LUCAS TM CPR. While none of the animals in the S CPR group survived, five of six animals in the LUCAS TM CPR group achieved ROSC and lived until euthanization two hours later. The following values were significantly higher in the animals receiving LUCAS TM CPR as compared with the animals receiving S CPR: diastolic pressure, mean arterial pressure, myocardial perfusion pressure, and coronary artery perfusion pressure. I n addition, after five minutes of CPR, LUCAS TM CPR generated significantly higher cardiac output, carotid arterial blood flow and coronary perfusion pressure. Interestingly, the five surviving pigs did not have significantly different arterial pressure,

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18 c arotid flow, or blood gas values from their baselines. In the Group II pig model, all pigs received 30 minutes of LUCAS TM CPR. Animals achieving ROSC were compared to animals that did not achieve ROSC. Coronary perfusion pressure and end tidal CO 2 valu es were similar for both groups at 5 and 15 minutes of LUCAS TM CPR. After 25 minutes of LUCAS TM CPR, the ROSC group had significantly higher levels of coronary perfusion pressure, end tidal CO 2 and carotid arterial blood flow, but significantly lower l evels of PvCO 2 In the Group III pig model, animals achieving ROSC after at least 15 minutes of LUCAS TM CPR were compared with pigs that did not have ROSC. Animals with ROSC had a greater mean coronary perfusion pressure and end tidal CO 2 than the non R OSC group. In the Group IV pig model, normothermic animals receiving LUCAS TM CPR were compared with animals that underwent surface cooling to achieve hypothermic body temperatures while LUCAS TM CPR was given. The normothermic animals had lower coronary perfusion pressure and greater metabolic acidosis than the hypothermic animals. In the clinical pilot study, 20 subjects received LUCAS TM CPR when S speed of application. One sub ject out of 20 survived to hospital discharge and had normal neurological function one year later. 12 AutoPulse A clinical study of a load distributing band (LDB) device, the AutoPulse, had a surprisingly negative outcome After promising studies in an animal model, Hallstrom et al. launched the AutoPulse Assisted Prehospital International Resuscitation (ASPIRE) trial. Five sites in the United States and Canada equipped ambulances with the AutoPulse. Patients located in pre determined geographical clusters that experienced cardia c arrest due to a likely cardiac cause were randomized to either the LDB CPR

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19 group or the S CPR group. Researchers operated the study under an emergency exception from informed consent in which each community involved in the research was publicly notified of the study. Individual subjects surviving to the follow up period provided informed consent for collection of outcome data. Rescuers had three different was quickly recorded in less than six seconds, and then they administered either LDB CPR or S CPR, depending on the randomization. After two minutes of CPR, rescuers assessed the rhythm. In the second option, rescuers immediately began S CPR until the firs t shock assessment. After the shock assessment and administration (if required), rescuers implemented the randomized CPR treatment. In the third option, began the randomize subsequently followed until ROSC or death was declared. The study proceeded for eight months before a data and safety monitoring board stopped it due to potential harm to participants undergoing the intervention. Among all sites, 1377 patients were enrolled in the study. Subjects in the LDB and S CPR groups had similar survival rates four hours after the initial 911 call. The LDB CPR group actually had lower survival rates to hospital discharge ( 5.8%) than the S CPR group (9.9%, p=.04). In addition, the S CPR group had a significantly better neurological performance at hospital discharge than those receiving LDB CPR. Although the researchers could not explain why the AutoPulse produced negative outcomes, they hypothesized that rescuers were unable to activate the device quickly enough, which may indicate that early blood flow in cardiac arrest is essential for survival. 13

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20 A dhesive G love D evice Our group developed an adhesive glove device (AGD) for active compression decompression CPR (ACD CPR). This device consists of a glove worn by the rescuer with a Velc ro patch sewn onto the palmar area of the glove. An opposing Velcro patch is adhered to the chest of the victim. This AGD assists rescuers in complete decompression of the chest. When the rescuer utilizes excessive decompression, the glove detaches fr om the Velcro adhered to the chest wall. Udassi et al. conducted a study in a pediatric manikin model to assess the efficacy of the AGD in ACD CPR compared to S CPR. AGD CPR and S CPR resulted in similar compression depths and pressures. Rescuers using t he AGD decompressed the chest significantly more than rescuers performing S CPR. While AGD CPR enabled rescuers to decompress the chest beyond the baseline level, few rescuers were able to decompress the chest to the baseline when using S CPR. Likely due to this greater decompression, rescuers administered fewer chest compressions while using the AGD as compared to rescuers in the S CPR group. Remarkably, all rescuers reported similar rates of fatigue, regardless of the type of CPR performed. In additio to baseline at a similar rate. 3 Microspheres To demonstrate the efficacy of CPR devices, research tools are required that illustrate blood flow to essential body organs. Researchers have used microspheres for several decades to evaluate regional blood flow to organs. Ini tially, radioactive microspheres were used. However, the processing of radioactive microsphere samples is involved and dangerous. Recently the BioPhysics Assay Laboratory (BioPAL TM Inc

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21 Worcester, MA), developed safer non radioactive stable neutron acti vated isotope microspheres. All microspheres follow the same general principle. The microspheres must be small enough to circulate in the body but large enough to be trapped in the capillaries of organs. Generally microspheres 15 m in diameter are inje cted. They are usually injected into the left atrium which is as far away as possible from the first major arterial branching. Injection into the left atrium ensures that the microspheres completely mix with the blood before branching to the organs. Bl ood flow to the organs is determined using a formula described by Heymann et al. 14 Commonly, researchers draw a reference blood sample at a steady rate as a type of organ. Researchers can then take the microspheres per gram of the organ of interest Reinhardt et al. validated a neutron activation assay technique using microspheres labeled w ith stable isotopes. Such microspheres are much safer to work with because they do not contain radioactive elements. In addition, tissue preparation is much faster because their assay does not require digestion of the tissue of interest prior to analysis Reinhardt et al. injected both stable labeled and standard radiolabeled 15 microspheres into a rabbit model of myocardial ischemia and reperfusion. After euthanasia, the researchers harvested the left ventricle and sliced it so each sample

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22 contained a pproximately equal portions of epicardium and endocardium. They also took samples from the brain, spleen, pancreas, stomach, small and large intestines, kidneys, and lungs. The stable labeled and radiolabeled microspheres indicated similar levels of bloo d flow (r=0.95 0.99) to the myocardium and all other organs. 15 CPR researchers use stable isotope labeled microspheres to illustrate blood flow to the organs before cardiac arrest and during various CPR techniques in animal models. Zuercher et al. used microspheres to demonstrate the effects of leaning on the chest on cardiac output during CPR. Researchers induced ventricular fibrillation in ten farm piglets. Nonradioactive microspheres (15) were inj ected into the left ventricle 30 seconds after the start of CPR. An automated withdrawal pump drew blood from the ascending aorta for 2 minutes and 30 seconds at a rate of 10 mL /min. Various forces were applied to the chest in three minute increments to chest compression. Microspheres of varying colors were injected during each three minute increment. None of the animals were defibrillated or achieved ROSC as this was not the goal of the study. Researchers removed 5x5 mm midanterior left ventricular wall tissue for microsphere analysis. Each sample was separated into endo and epicardial sections. Ten and twenty percent leaning produced similar values of blood flow. However, when full recoil of the chest was allowed, si gnificantly more blood flow was noted, likely due to the generation of negative intrathoracic pressure. 16 Bioma rkers The low blood flow state of cardiac arrest and the onslaught of cytokines during brain reperfusion can lead to neurological damage. A victim of cardiac arrest does not manifest the full consequences of cardiac arrest for several months after neurolog ical insult. Biomarkers released from damaged neurons may help physicians identify the

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23 likely extent of neurological damage well before a patient achieves a full recovery. Unfortunately, a single biomarker will not give the most accurate prediction. An entire neurological function. Neuron specific enolase and S100 B are two such biomarkers that have previously been studied. In this study, we investigated three potential bio markers: Ubiquitin C terminal Hydrolase 1 (UCHL1), phosphorylated neurofilament type H (pNF H), and alpha syn). UCHL1 and pNF H Lewis et al. developed an assay for detection of UCHL1, a marker of perikarya and dendrite destruction. The researchers soaked bovine cerebral tissue in calcium salts to which protein s would be abundant and easily released into the CSF. They then used SDS PAGE to separate the proteins. The bands at 24 kDa were then excised and underwent tandem mass spectrometry, which revealed the presence of UCHL1. Western blotting showed that the b ands were full length UCHL1. UCHL1 is present in the perikarya and dendrites of neurons and in neu roendocrine cells. Lewis et al. developed an enzyme linked immunosorbent assay (ELISA) f or detection of UCHL1. They identified a monoclonal antibody that wo uld bind UCHL1 of rats, humans, and cows. This ELISA consisted of plates bound with a monoclonal antibody to UCHL1 ( BH7 ) that were bound to rabbit anti UCHL1, that were bound to anti rabbit goat alkaline phosphatase conjugate and then incubated with p ni trophenol chromagen. The researchers subsequently used their ELISA for UCHL1, along with assays for S100B (a well established biomarker) and pNF H (another biomarker in the testing phase), to analyze CSF samples collected daily from ventriculostomies of a dult patients with

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24 aneurysmal subarachnoid hemorrhage (ASAH). R esearchers successfully generated UCHL1 antibodies for rabbits, chickens, and mice that reacted in crude brain samples, stained neuronal perikarya, and concentrated protein samples. Researcher s discovered that CSF samples from healthy control subjects did not contain any UCHL1 and minimal pNF H (<0.89 ng/m L ) and S100B (<0.55 ng/m L ). UCHL1, pNF H, and S100B levels were elevated in all CSF samples from ASAH subjects. In addition, subjects with the worst outcomes had the highest biomarker levels. Biomarkers UCHL1 and pNF H were present 24 hours after hemorrhage and were still being released 14 days after ASAH, while S100B levels decreased 4 days after ASAH. The S100B levels likely decreased qui ckly because S100B is associated with astrocytes, which are hardy cells that only break down during the acute rupture of the aneurysm and periods of extreme hypoxia. Researchers attributed fluctuations in S100B levels to vasospasm. UCHL1 and pNF H likely stayed elevated for a longer period of time than S100B because they are released from neurons, which are more susceptible to trauma from transient ischemia and secondary neuronal cell death. 17 Biomarkers such as S100B are found in both the central (CNS) and peripheral nervous systems (PNS). However, the extent of contribution from the PNS to the serum level of S100B is unknown. UCHL1 is also found in the CNS and PNS, but UCHL1 is 50 times mo re abundant in the CNS when compared to the PNS. pNF H is only in neurons of the central nervous system. S100B, UCHL1 and pNF H are found in the CSF and in plasma. Due to the abundance of these biomarkers in the CNS and in the plasma, these biomarkers mu st cross the blood brain barrier (BBB).

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25 Blyth et al. compared 16 patients with TBI to 6 patients that underwent lumbar puncture for extreme headaches. Subjects with TBI had blood drawn and CSF taken every 12 hours after injury up to 48 hours. The headac he patients had blood drawn at the time of lumbar puncture. S100B, UCHL1, and pNF H levels were compared to the Q A an assessment of BBB permeability that compares albumin in the CSF and plasma, to determine a correlation between biomarker levels and BBB function. S100B and UCHL1 levels as well as Q A increased 12 hours after injury when compared with the headache controls. This increase indicates a breakdown of the BBB. pNF H did not increase above the headache controls at any point in this study (12 4 8 hours after injury). The researchers hypothesized that pNF H levels were not increased because pNF H is a large protein, which would require significant BBB dysfunction. This dysfunction would most likely occur immediately after TBI, which was not exam ined in this study. In addition, pNF H is found mostly in the white matter which is more prevalent in the spinal cord. This study looked at damage in the cortex, which has more gray matter than white matter. 18 Alpha synuclein Another biomarker, alpha syn) is found in the plasma and CSF of syn levels are altered in patients with dementia, e. 19 21 Su et al. syn levels in CSF from children with traumatic brain injury (TBI) to CSF obtained from children suspected of having an infection but later deemed healthy. The researchers also randomized the subjects with TBI to receive either the standard of care or neuropr otective therapeutic hypothermia. Su et al. discovered that subjects with TBI syn levels were higher in

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26 subjects with TBI due to non accidental trauma than subjects with accidental trauma. This fi nding was likely due to more severe neuronal damage in TBI due to non accidental trauma. Those subjects treated with therapeutic hypothermia had lower syn than subjects receiving the standard of care. The researchers hypothesized that this re syn was either due to less inflammation and neurotrauma in subjects treated with therapeutic hypothermia or due to protein transporters and exocytosis slowed from the decrease in temperature. For unknown reasons, females in the TBI grou syn. In addition, subjects under syn. This finding was probably due to increased syn levels were seen in days 4 6 after TBI when secondary neurotr auma frequently occurs. The researchers could not syn release from the presynaptic areas. 22

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27 CHAPTER 3 METHODS We conducted a randomized controlled trial of an AGD for use in ACD CPR in a swine model. Control animals received S CPR. The University of Florida (UF) Institutional Animal Care and Use Committee approved this experimental protocol. Qualified animal personnel performed all interventions under the supervision of a veterinarian and followed the guidelines of the American Physiological Society. Methods are reported according to the Uts tein guidelines. 23 Animal Preparation Thirty one farm pigs (2 months old, 11 19 kg, UF Swine Unit) of either gender were sedated with ke tamine (15 mg/kg, IM injection) and inducted with isoflurane (3 5%) until anesthetized. Anesthesia was maintained with isoflurane (0.5 1.5%). Intravenous catheters (20 24 g) were inserted bilaterally into each ear vein. The pigs received propofol (2 4 m g/kg, IV) as needed for intubation. The animals were then intubated using cuffed 5 6 mm endotracheal tubes. Mechanical ventilation was maintained with a rate and volume regulated ventilator (SurgiVet Vaporstick Anesthesia Machine, Smiths Medical, Norwe ll, MA). Initial ventilation was set at 12 respirations per minute with a tidal volume of 15 mL /kg. Rate and tidal volume were adjusted to maintain an end tidal pCO 2 of approximately 40 mmHg. The lowest concentration of anesthetic that prevented movement during surgical instrumentation was used. EKG leads were placed on the limbs to continuously monitor heart rate and rhythm.

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28 Surgical Procedures The common carotid arteries, left external jugular vein (EJV), and femoral arteries were surgically exposed fo r instrumentation. A 3mm T206 flow meter (Transonic Systems Inc, Ithaca, NY) was situated around the left common carotid artery. A 5F vascular introducer sheath was placed in the left EJV and advanced to the upper portion of the right atrium. A fluid f illed catheter transducer was inserted into this introducer for right atrial pressure measurement. In this same introducer, using fluoroscopy for guidance, a non coated guide wire was inserted into the EJV and advanced to the right ventricle for future in duction of ventricular fibrillation. A 6F vascular introducer sheath was placed in the left femoral artery. Through this introducer sheath, a 6F pigtail catheter (Cook Medical, Bloomington, IN) was advanced to the left ventricle for stable isotope lab eled microsphere injection (BioPAL, Inc, Worcester, MA). Fluoroscopy was used to confirm placement. A 7F double lumen catheter (Cook Medical, Bloomington, IN) was placed in the right femoral artery for blood pressure monitoring and blood collection. A Mikro Tip pressure catheter (Millar Instruments Inc, Houston, T X ) was inserted into the 4th intercostal space on the right. Catheter data were acquired using a PowerLab data acquisition system and LabChart 7 (ADInstruments, Sydney, AU). Adhesive Glov e Device The adhesive glove device (AGD) consisted of a golf glove with the fingers removed. A Velcro (3M TM St. Paul, MN) patch was sewn onto the palmar aspect of stit ched in place using surgical sutures

30 Ventricular Fibrillation Ventricular fibrillation (VF) was induced via direct right ventricular current in all sedated pigs. VF was confirmed with EKG waveform and a sharp de crease in aortic pressure. To simulate a real life cardiac arrest situation, assisted ventilation was discontinued and no rescue interventions were performed for 3 minutes. An interim analysis indicated that 3 minutes of VF was not long enough to show a difference between groups. As a result, animals induced towards the end of the study were allowed to stay in VF for 5 minutes. Thirty seconds before the end of the VF period, arterial blood for inflammatory marker analyses was drawn. Resuscitation After 5 minutes of untreated ventricular fibrillation, the pigs received either S CPR or AGD CPR for two minutes by rescuers blinded to data recording. The compression rate was 100/min with a compression to ventilation ratio of 30:2. During these first two min utes of CPR, blood was drawn and 2.2 mL (5.5 million particles) of gold microspheres was injected for blood flow analysis (BioPAL, Inc, Worcester, MA) as described above. The pigtail catheter used for microsphere injection was removed to prevent damage to the left ventricle during chest compressions. CPR was carried out according to Pediatric Advanced Life Support guidelines (American Heart Association, Dallas, TX) until return of spontaneous circulation (ROSC). ROSC was defined as an organized heart rh ythm with a systolic aortic BP greater than 50 mmHg and a MAP greater than 20 mmHg lasting at least one minute. Venous and arterial blood gases and electrolytes were monitored 30 minutes after ROSC. Arterial blood (3 mL ) for biomarker analysis was collect ed at ROSC, 30 and 90 minutes after ROSC, and

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31 immediately prior to euthanasia. Surviving animals were monitored for 4 hours after ROSC and then euthanized with Beuthanasia (Merck, Summit, NJ) Necropsy All animals underwent gross dissection to investigate possible thoracic cavity trauma. In addition, a 1 g piece of the following tissues were obtained for microsphere analysis: cerebral cortex, kidney cortex, liver, and apex and interventricular septum of the heart. Microsphere Processing Vials of blood obtained during microsphere injection were centrifuged for 1 minute at 2,000 RPM. The supernatant was then aspirated to a level safely above the visible pellet. The pellet was re suspended in sanSaLine TM an electrolyte free saline, (BioPAL, In c, Worcester, MA) and re centrifuged and aspirated. Tissues obtained for microsphere determination were rinsed with sanSaLine TM weighed, and inserted into a tissue sample vial. SanSaLine TM removes interstitial sodium and chloride and surface blood to he lp reduce the signal to noise ratio of the analysis. The vials of blood and tissue were dried in a warming oven overnight at 70 degrees Celsius. This step dried all liquids to prevent spillage and contamination during ship ping All samples for each subj ect were placed into a single sealed bag and labeled. With the sample vials at room temperature, an assay request form stating the duration of blood draw and wet weight of each tissue sample was mailed via FedEx to BioPAL, Inc. BioPAL performed all micro sphere analyses and blood flow determination as previously published. 14,15

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32 Biomarker Sample Processing Blood for biomarker sampling was allowed to clot in a serum separator tub e, and then centrifuged at 3200 RPM for 10 minutes. The serum (supernatant) was removed, placed in an Eppendorf tube, and stored at 80 C The clotted red blood cells and blood collection tube were appropriately discarded. At the end of the study, all s erum samples were transported to the EnCor Biotechnology, Inc. (Gainesville, FL) laboratory in a cooler packed with ice. The EnCor laboratory performed assays for determination of biomarker levels as previously described. 17,24 26 Statistical Analysis Continuous variable descriptive data were analyzed using an unpaired t test and are p resented as means ( standard deviation). To determine differences, categorical variables were analyzed by Chi Square and are presented as proportions. A Kolmogorov Smirnov test and inspection of error residuals tested data for normality. Repeated measu res ANOVA models compared mean differences of biomarker value measurements over time. In addition, the repeated measures ANOVA was used to determine if the biomarker values in the S CPR group interacted with the biomarker values in the AGD CPR group over time. Post hoc comparisons for respective time points to baseline were performed using Dunnett Hsu correction, with P < 0.05 considered significant. Unpaired t tests assessed mean differences between animal groupings for microsphere organ blood flow asse ssments and at a given time point for biomarker values. Data were analyzed using SAS V9.2 (SAS Institute, Cary, NC, 2012).

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33 CHAPTER 4 RESULTS Laboratory Blood Parameters S CPR and AGD CPR groups were similar at baseline (Table 4 1). Blood gases and electrolyte values taken at baseline and after resuscitation were similar for S CPR and AGD CPR groups. At baseline, the AGD CPR group had significantly higher potassium, venous bicarbonate (HCO 3 ) and base excess levels; however these elevated levels wer e still within a normal clinical range. Thirty minutes after ROSC, blood gas and laboratory parameters for S CPR and AGD CPR groups were still similar. Although potassium and ionized calcium means were significantly higher in the AGD CPR group, these lev els were within the normal clinical range (Table 4 2). Non radiolabeled stable isotope microspheres Six animals from the S CPR group and six animals from the AGD CPR group underwent microsphere analysis to determine regional blood flow. Results from all o rgans analyzed varied widely The S CPR and AGD CPR groups did not have significantly different blood flow to the brain, heart apex, heart septum, kidney, or liver. In addition the S th at was greater than the mean. The AGD flow had a standard deviation that was almost as great as the mean. Mean blood flows for the heart apex, heart septum, kidney, and liver for both groups exhibited similar wide s tandard deviations (Table 4 3) Biomarkers UCHL1, pNF syn were formally tested for interactions between time and CPR group. UCHL1 ( P syn ( P = 0.62) and pNF H ( P = 0.95), did not

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34 demonstrate observable interactions. Interestingly, al l biomarkers moved in a similar fashion over time ( F igures 4 1, 4 2, and 4 3). Because the interactions between time and CPR group were not significant, the main effects of biomarkers drawn at individual time points after induced ventricular fibrillation w ere compared to the respective baseline. In addition, the S CPR and AGD CPR groups at given time points were compared to each other ( T able 4 4 ). UCHL1 At baseline, UCHL1 levels were similar for S CPR and AGD CPR. In addition, UCHL1 levels clustered near the mean value (Figure 4 4 ). The UCHL1 markers at each time point were not significantly different than the baseline when compared using the repeated measures ANOVA (S CPR, P =0.54; AGD CPR, P =0.65). UCHL1 markers drawn at ROSC and pre euthanasia in the S CPR group appeared higher than the baseline, but were not significantly greater (ROSC P =0.17,pre euthanasia P =0.70) (F igure 4 1 ) Alpha synuclein At baseline, the S syn level (6.15 ng/mL ) than the AGD CPR group (1.21 ng/mL P = 0.05) (Figure 4 2) However, the standard deviations for both S CPR (7.72) and AGD CPR (1.25) groups at baseline were notably higher than the means (Figure 4 5) At baseline, the S CPR group had pronounced deviations from the mean (6.14 7.72 ng/mL syn due to outliers of 13.94, 14.07, and 25.54 ng/mL Figure 4 3 displays the pronounced effect of outliers at baseline, during ventricular fibrillation, and at 30 minutes after ROSC. Without including the outlying values at baseline, t syn S CPR baseline mean (2.24 1.51 ng/mL ) would be similar to the AGD CPR group ( P = 0.07).

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35 The repeated measures ANOVA models for S CPR ( P = 0.85) and AGD CPR ( P = 0.78) were non significant for time. In the S syn markers drawn at all of the time points, except at two minutes of CPR, deviated very little from the baseline level. However, only three samples were able to be drawn at two minutes of CPR due to insufficient blood pressure ( F igure 4 2 T able 4 4 ). In the AGD CPR group, the syn markers drawn at all of the time points were similar to the baseline level (Figure 4 2 ). pNF H As with the previous biomarkers, pNF H levels did not have significant differences between the means with regards to time in either the S CPR group ( P = 0.51) or the AGD CPR group ( P = 0.75). The S CPR group had an initial mean baseline value of 0.095 ng/mL (Figure 4 3 Table 4 4 ) and remained steady throughout all subsequent time points. In contrast, pNF H levels in the AGD CPR group displayed a rathe r erratic deviation from the baseline. However, none of these deviations from baseline were statistically significant. In addition the S H levels were not significantly different from the AGD CPR group levels. Time Main Effect A sub analys is combined S CPR and AGD CPR groups and compared the effect of time in order to see how the population acted as a whole. Marker UCHL1 appeared to be at a steady state, slightly decreased after two minutes of CPR, rose, decreased 90 minutes after ROSC, th en rose again pre euthanasia (Figure 4 7). Notably, few observations occurred at two minutes of CPR and at 90 minutes after ROSC. With these scant observations omitted for marker UCHL1, a steady line would extend from the baseline, rendering the model no n significant ( P = 0.79).

44 CHAPTER 5 DISCUSSION The portion of this study presented in this manuscript did not show a difference between AGD CPR and S CPR. This homogeneity can be attributed to physiological, statistical, and methodological factors. Our study started with two similar groups of healthy young pigs. At the end of the study, both groups of animals had electrolyte values and blood gases within the normal range. This similarity of electrolytes between groups is not surprising as all animals underwent aggressive resuscitation and were supported as needed with an epinephrine drip after ROSC. I n addition, animals were given the same amount of recovery time (30 minutes) after ROSC on controlled ventilator settings before their blood was drawn for electrolytes and blood gases. In order to elucidate a difference between CPR groups in the future, we could best illustrate the immediate effects of S CPR versus AGD respiratory function and acid base balance. In addition, we could continue ventilat ing the animals mechanically after ROSC but not assist the weaker animals with epinephrine. The non radioactive labeled isotope microspheres were not helpful in determining regional blood flow. In a prior animal study conducted by our lab (unpublished), t he same microsphere protocol was followed as in this study. Animals receiving AGD CPR had significantly higher blood flows to organs than in S CPR. The sample size for this prior study was small (n=16) and likely rendered a falsely significant result. T his Type II error was not visible to our team until the results of the current study were analyzed.

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45 We committed several errors in carrying out the microsphere protocol. First, we used a catheter with too many lumens connected to a long extension tube dur ing a low flow state. As a result, the triple lumen catheter required extensive manipulation at the time of blood draw. Because our stopwatch was started at the same time as the syringe withdrawal pump, this delay caused less blood to be drawn over the c ourse of two minutes. This parameter change made the blood flow calculation done by BioPAL, Inc inaccurate. After experiencing difficulty with blood flow through the triple lumen catheter during CPR with several animals, we began using a double lumen cat heter. In addition, we moved the syringe withdrawal pump closer to the animal and omitted the long extension tubing in order to reduce the distance the blood had to travel. In the future, we should wait to start the two minute stopwatch until the blood i s definitely being withdrawn. We could also weigh the withdrawn blood in a pre measured vial to determine the quantity of blood withdrawn. We made a second microsphere error in collecting the tissues post mortem. Although we collected tissue from the sam e organ area in each animal, the tissue samples obtained were probably too small to give a good representation of the entire organ. Researchers in similar cardiac arrest studies sampled several pieces of an organ and sectioned the samples into layers. In the heart, we collected the entire apex of the heart and a piece of the intraventricular septum. Lurie et al. collected a portion of the left anterior ventricle and separated it into three layers for analysis. 27 Zuercher et al. focused on the mid anterior free wall of the left ventricle and separated the epicardium from the endocardium. 16 In a healthy heart, the endocardium usually receives more blood flow than the epicardium. However, in cases of ischemia the

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46 opposite is true: the epicardium receives more blood flow than the endo cardium. 28 During S CPR the blood flow in the heart would likely be similar to a state of ischemia. However, AGD CPR can be very aggressive and generate high rates of blood flow, mimicking a healthy heart. Because we were comparing S CPR to AGD CPR in this study, we should have collected a specimen from the left ventricular wall and separated the epicardium from the endocardium. Then, we could compare the heart layers from the animals in each CPR group. Microspheres are a well documented tool. Beginning with radioactive microspheres in the 1970s, researchers have extensively used them to determine blood flow rates. 14 With the advent of non radioactive neutron activated microspheres in 2001, this technique is much safer. The label of the isotope is cross linked with the polystyrene microsphere. As a result, the isotope is highly unl ikely to leak from the nanoparticle even under extreme temperature and pH conditions. 15 Although we were unable to show accurate blood flow in our current study, microspheres are still a valid tool that s hould not be abandoned. If we refine our technique, neutron activated microspheres may be helpful in showing differences between S CPR and AGD CPR. While microsphere blood flow analysis is well validated, biomarkers for neurologic injury during cardiac ar rest are fairly new. Our study indicated the presence of biomarkers pNF syn in the plasma of swine before and after cardiac arrest. However, the fluctuation of biomarkers rendered our study inconclusive with regards to neurological injury Several factors might have caused fluctuation of biomarker levels. First, biomarker detection relies on appropriate timing of blood draws. Many studies look at

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47 biomarkers in chronic neurodegenerative diseases such as amyotrophic lateral sclerosis, Park 25,29,30 As a result, the exact timing of neurological damage in these diseases is unknown. In addition, several studies examining biomarkers after acute events such as a stroke traumatic brain injury, and cardiac arrest draw blood 12 to 48 hours after the event, largely due to a 17,18,31 Because we had the occurring, we drew blood for biomarkers before, during, and immediately after the neurological insult. Unfortunately, we do not kno w if the marker levels we observed are after injury. To determine the exact amount of time required for neurological damage from ischemia to manifest in the plasma, we would need to draw blood for a baseline level, simulate direct neurological ischemia, then draw blood every 15 minutes for several hours. An even more direct method of determining the release of biomarkers would be to perform a ventriculostomy to withd raw CSF in regular intervals and draw blood concurrently. This method would detect the time of biomarker release into the CSF as well as the time the biomarkers cross the BBB. The BBB might also cause biomarker fluctuation. An intact BBB prevents the passage of most proteins. During cardiac arrest, the BBB becomes permeable as early as five minutes after cardiac arrest. The most significant increase in permeability occurs 30 minutes after cardiac arrest and progressively continues for at least 3 hours. The BBB breaks down first in the hippocampus, cortex, and hypothalamus. When this breakdown in the BBB occurs, proteins can move freely from the plasma into the CSF

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48 and from t he CSF into the plasma. This event causes an influx of cytokines to enter the brain tissues. As a result, edema and ischemia can occur within the brain. This edema and ischemia can damage axons, neurons and glia. 32 If the biomarkers we tested for only came from the CNS, we could say th at the presence of biomarkers in the blood meant that the BBB had become more permeable and damage to the brain occurred during cardiac arrest. However, we cannot verify that two of the biomarkers we tested only came from the brain. UCHL1 is mainly found in the brain, but other tissues outside the CNS have stained for UCHL1. These tissues include the distal renal tubules, the pancreas, and the testis. Alpha synuclein is present in the CSF and plasma of normal individuals. Thus, we cannot be certain that syn found in the plasma of our study has any significance as a biomarker of brain injury. syn, pNF H is only found in the brain. The earliest significant increase in pNF H in a murine model was eight hours after brain injur y. 24 Our data did not show a significant increase in pNF H, however we only drew blood up to four hours after brain injury. W e did note the presence of pNF H in the plasma. If we had continued drawing blood for several more hours, we might have seen a significant rise in pNF H levels. Interestingly, researchers have reported that the highest levels of pNF H appear in the spinal cord and brain stem, followed by the cerebellum, thalamus, and putamen. The lowest levels of pNF H were detected in the hippocampus, cerebral cortex, and campus callosum. 33 As mentioned above, the BBB breaks down first in the hippocampus, cortex, and hypothalamus, which coincidentally are areas with the lowest levels of pNF H during cardiac arrest. 32 This variability in BBB breakdown versus

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49 axonal breakdown may cause the fluctuating levels we found in th e plasma. A specific brain region might have axonal breakdown, but the biomarker might not be released immediately into the plasma because the BBB in the given brain region is still intact. Thus, pNF H may not be detectable in the blood immediately after cardiac arrest, but could be present in the CSF. In the future, our study would need to continue sample collection for several hours to several days in order to observe a difference in cardiac arrest between groups in regards to biomarkers in the blood. In addition to collecting biomarkers for a longer duration after cardiac arrest, the duration of cardiac arrest in our model should be increased. Initially we allowed the animals to remain in untreated cardiac arrest for three minutes. After an interim a nalysis, we increased the duration of untreated cardiac arrest to five minutes. This time was obviously not long enough to show a significant difference between groups. Other researchers that have worked with biomarkers in cardiac arrest have allowed as long as 12 minutes of untreated cardiac arrest before resuscitation. 32 Notably, significantly more animals in the AGD CPR group (95%) survived than in the S CPR group (67%, P=0.04) in the post arrest period. However, within 30 minutes of resuscitation 2 of the animals in the AGD CPR group died, which re duced the significance of this variable (P=0.25). Blood flow, laboratory values, and biomarkers are all important factors that should contribute to chances of survival. However, the w many lives are saved and the neurological ability of the patients that survive. This data is the strongest factor in determining that the AGD works in improving the chance of survival of cardiac arrest. According to Utstein style guidelines, true decla ration of survival of cardiac arrest

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50 neurological status, detection of multisystem failure, and assessment of cardiovascular physiology following withdrawal of pharmacocirc 23 Our group plans to carry out a survival study with neurological assessments in the near future. Previously, o ur group showed that the AGD improves decompression during CPR and does not cause additional fatigue to rescuers. 3,34 In order to prove th at the AGD improves survival, we must carry out a study with greater statistical power. This study was too severely underpow ered to show any differences. F or example, in a post hoc power analysis, the best power from the microsphere data was from blood f low to the kidney with a power of 6.5%. Unfortunately, animal studies of this scale are very expensive. The average cost per pig procedure of this study was $2,000. While this study is underpowered, it was instrumental in illustrating changes that need to be made to improve the protocol. This study also showed that our group is capable of a cardiac arrest swine model, which is necessary to secure additional grant funding.

55 BIOGRAPHICAL SKETCH Melissa Lamb graduated Phi Beta Kappa from the University of North Dakota with a Bachelor of Arts in Music in 2002. She then pursued two years of medical school at the University of North Dakota. From 2005 to 2011, Melissa worked as a research coordinator in Pediatric Critical Care at the University of Florida. Her work included basic science, translational, and clinical research in the subjects of neurology pulmonology infectious disease, and cardio logy. Melissa currently works as a free lance medical editor and scientific writer.